Proteolytic enzymes in urine as diagnostic parameters in diseases involving matrix remodelling

ABSTRACT

A method for determining whether a urine sample has been obtained from a subject suffering from a kidney disorder involving kidney damage. The method is based on determining the level of a proteolytic enzyme in urine from the subject. Sample and optionally comparing this level with a reference value. The proteolytic enzyme preferably is a matrix metalloproteases, in particular MMP-2 or MMP-9. The method is particularly suitable for subjects with a condition or disorder that is associated with an increased risk of kidney disorders, such as diabetes. The method may adavnatageously be applied for early detection of kidney damage, in particular when the subject does not yet show any sign of microalbuminuria. The method may also be applied to a subject undergoing dialysis for determining whether the subject is suffering from remodelling of the peritoneal membrane. Thus, the method is particularly suited for patients undergoing continuous ambulatory peritoneal dialysis.

FIELD OF THE INVENTION

This invention is in the field of methods and techniques for theprediction and/or diagnosis of renal disorders, and in particular forthe prediction and/or diagnosis of renal damage associated withdiabetes, and in particular with diabetes mellitus. In particular, theinvention relates to a method of determining the presence and amount ofa proteolytic enzyme in urine and the use thereof as diagnostic orprognostic parameter in diseases involving matrix remodelling such asdiabetes.

BACKGROUND OF THE INVENTION

Increased or decreased matrix remodelling is involved in many diseasesand pathological processes, including cancer, angiogenesis, restenosis,arteriosclerosis and other vascular remodelling processes, vascularcomplications of diabetes such as retinopathy and nephropathy.Dysregulation of remodelling processes frequently causes problems ormight be even essential in the development of the particular disease.Matrix remodelling plays a key role in invasion and metastasis of tumorcells, cartilage degradation in rheumatoid arthritis and vascularremodelling. Proteolytic enzymes belonging to the plasmin-system and thematrix-metalloprotease system are main actors in the various remodellingprocesses. Consequently measuring these components or influencing theiractivity are potential targets for diagnostic or therapeutic methoddevelopment.

Vascular complications and renal disease are frequent complications ofe.g. diabetes mellitus. Early renal disease is accompanied by leakage ofprotein into the urine (microalbuminuria) which is used as a diagnosticmarker for this complication. Microalbuminuria appears to be preceded bywidespread endothelial dysfunction, therefore much attention has beendevoted to the study of the possible use of circulatingendothelium-derived molecules as markers of early stage diabetic kidneyfailure. Local proteolytic activity is involved in micro-vascular damageleading to kidney failure and measurement of the local level or activityof certain proteolytic enzymes or their inhibitors might be indicativeof occurring damage to microvascular integrity. Some data have beenpresented suggesting that increased levels of matrix metalloproteases,one group of proteolytic enzymes thought to be involved in formation ofvascular damage, occur in peripheral blood possibly as a representationof local levels in the kidney.

For the above, reference is inter alia made to I. Massova et al., FASEBJ. 12, 1075-1095 (1998); P. Primakoff et al., Trends Genet. 16, 83-87(2000); and B. L. Tang and W. Hong, FEBS Lett. 445, 223-225 (1999).However, none of these references discloses or suggests to determineproteolytic enzymes in urine samples.

Generally, the measurement of proteolytic enzymes in periferal blood,serum or plasma is cumbersome, as it involves the use of blood samples,which have to be collected from the patient by a docter or a skilledmedical technician. This may put a great deal of strain on the patient,not only because of the invasive techniques required to collect theblood sample, but also because this will usually involve a visit to aclinic or a hospital. In addition blood, serum or plasma containnumerous compounds potentially interfering with the measurement ofproteolytic enzymes requiring carefully developed procedures which areprone to complications. In urine hardly any interfering substancesoccur, thus making reliable measurement of proteases much more simple.

Lenz et al., Journal of the American Society of Nephrology, March 2000,Vol. 11, No. 3 review the role of matrix metalloproteinases in renaldevelopment and disease, including non-inflammatory glomerular diseasessuch as diabetic nephropathy.

However, in doing so, Lenz et al. refer either to in vitro studiesinvolving the measurement of MMP-expression and -activity at thecellular level (e.g. in mesangial cells), to studies involving the useof kidney biopsies, or to studies involving the measurement ofexpression of MMP-encoding mRNAs. Thus, this reference does not suggestto measure MMPs in urine, and also does not teach that the levels ofproteolytic enzymes in urine can be used as diagnostic markers for(early) kidney damage associated with diabetes (mellitus).

Furthermore, with reference to the studies mentioned in their review,Lenz et al. teach that, contrary to inflammatory glomerular diseasessuch as glomerulonephritis, which are generally associated withincreased expression and activity of MMPs, MMP-expression andMMP-activity are in fact decreased in non-inflammatory glomerulardiseases such as diabetic nephropathy. In this respect, it should benoted that according to the invention, early renal damage associatedwith diabetes was found to be associated with a marked increase in thelevels/activity of—for example—MMP-2 and MMP-9 in urine.

Senatorksi et al., Res. Exp. Med (Berl) 1998 December; 198(4):199-206,describe a study in which urine activity of cathepsin B and collagenaseas well as urine excretion of TGF-β1 and fibronectin were measured inpatients suffering from membranous glomerulonephritis.

However, as stated in the first paragraph of the section entitled“Materials and methods” on page 201, this study specifically excludedpatients with diabetes. Thus, this reference teaches the skilled personnothing on the levels of proteolytic enzymes in the urine of patientssuffering from diabetes. In particular, this reference does not teachthat the levels of proteolytic enzymes in the urine of a diabetespatient can be used as diagnostic markers for (early) kidney damage.

In addition, Senatorksi et al. generally refer to the measurement of“collagenases” in urine, in which the term “collagenases” is apparentlyused generically to indicate any and all enzymes from the (veryextensive) entire group/family of enzymes that may degrade nativecollagen. This is confirmed by the fact that the fluorometric techniqueused by Senatorksi to determine said “collagenases” is not very specific(e.g. for individual collagenases or MMPs).

Also, two of the most important markers in urine used according to thepresent invention, MMP-2 and MMP-9, are not “collagenases” but“gelatinases” (compare the discussion of MMP-2 and MMP-9 in the reviewarticle by Lenz et al., mentioned above).

In the invention, it has now been found that the presence and/or thelevels of certain proteases in the urine of a subject may be used as adiagnostic and/or prognostic parameter for renal disorders, and inparticular for (early) renal damage associated with diabetes mellitus.

In particular, in the invention, it has been found that the presenceand/or the levels of certain proteases in urine may be indicative forrenal damage which is not—or not yet—detectable by the known method ofdetermining the leakage of protein in the urine. Thus, the method of theinvention is not only more reliable than determining (the extent of)microalbuminuria, but may also provide valuable information to theclinician even before any damage to the kidneys has already progressedto such a stage that microalbuminuria occurs.

Also, in the invention, it has surprisingly been found that the presenceand/or the levels of said proteases in urine may be used as reliablemarkers for (early) renal damage which is not—or not yet—detectable byincreased levels of matrix metalloproteases in peripheral blood. Thus,besides putting less strain on the patient and generally being moreconvenient, the method of the invention is also more reliable thanmeasuring the amount of proteases in blood or plasma, allowing forearlier detection of possible renal damage, in particular in diabeticpatients.

It should however be noted that the invention is not limited to (early)detection of renal damage in diabetes or other diseases with kidneyfailure as complication, but can also be employed for monitoring ordiagnosis of remodelling of the peritoneal membrane. Remodelling of theperitoneal membrane is a frequent problem in patients undergoingcontinuous ambulatory peritoneal dialysis (CAPD). For instance, one ofthe further (non-limiting) applications of the invention that isenvisaged is to monitor adequacy of dialysis in such patients.

SUMMARY OF THE INVENTION

The invention provides a method of using the determination of one ormore proteases alone or in combination, in urine as a sensitivediagnostic or prognostic marker for early kidney damage as e.g. oftenoccurring as a complication of diabetes mellitus. Said proteases couldbelong to the group of matrix metalloproteases or related enzymes suchas ADAMS or ADAMTS but also certain serine-, cysteine- oraspartyl-proteases could be used. Furthermore said proteases might befragments of originally larger proteins, complexes of proteins, or evensoluble fragments of normally insoluble, membrane bound, enzymes whichare still recognizable either by remaining enzymatic activity orantigenic determinants. Urine is a very convenient medium—since itnormally contains no or extremely low levels of proteolyticenzymes—which is frequently used in current diagnostic methods fordiabetes or diabetic nephropathy like glucose level, or albumin orprotein level and thus fits well with current clinical routine practice.

Thus, in a first aspect, the invention relates to a method fordetermining whether a urine sample has been obtained from a subjectsuffering from a kidney disorder and/or from kidney damage, said methodcomprising the steps of:

-   a) providing at least one urine sample from said subject;-   b) determining in said at least one urine sample the presence and/or    the level of at least one proteolytic enzyme;    and optionally comprising the further step of:-   c) comparing one or more of the values determined in step b) with at    least one reference value or reference sample.

The invention also relates to a method for detecting and/or monitoring(early) kidney damage and/or kidney disorders in a patient, comprisingsteps a) to c) above.

In particular, the urine sample provided in step a) may be a samplecollected from a patient/individual who is judged to be at riskof—and/or who is suspected to suffer from—a kidney disorder and/orkidney damage; or more generally from a patient that suffers from acondition or disorder that may cause and/or that is associated withkidney disorders and/or kidney damage or an increased risk therefor(e.g. as a complication of said condition or disorder).

More in particular, the urine sample provided in step a) may be a samplecollected from a patient/individual who is judged to be at riskof—and/or who is suspected to suffer from—kidney damage caused by and/orassociated with diabetes; or more generally may be a sample collectedfrom a patient suffering from diabetes.

Even more in particular, the urine sample provided in step a) may be asample collected from a patient/individual who is at risk of—and/or whois suspected to suffer from—kidney damage caused by and/or associatedwith diabetes mellitus type 1 and/or diabetes mellitus type 2; or moregenerally may be a sample collected from a patient suffering fromdiabetes mellitus type 1 and/or diabetes mellitus type 2.

However, the invention may also be applied to urine samples that havebeen obtained from patients that are suffering from and/or suspected tosuffer from other disorders that may lead to renal damage (including butnot limited to microalbuminuria) or an increased risk thereof, includingbut not limited to disorders such as (auto) immune mediatedglomerulonephritis, amyloidosis, nephrotic syndrome with various causes,paraneoplastic nephropathy, renal damage associated with multiplemyeloma or fibrosis in which it may be indicative or correlate with lossor decline of renal function. The invention may further be applied tourine samples that have been obtained from patients with organtransplantation, more particularly kidney transplantation, as an earlymarker for rejection of the transplanted organ.

In one particularly advantageous embodiment, the urine sample providedin step a) is a sample collected from such a patient/individual—and inparticular from a diabetes patient—who does not or not yet show anysign(s) of microalbuminuria. Usually, this means that the urine sampleused in step a) will have an overall protein content expressed asalbumin/creatine ratio (ACR)—e.g. as measured by standard methods suchas turbidimetry, nephelometry or immunodiffusion for albumin/proteincombined with determination of creatinine using well known proceduressuch as the method of Jaffé—of no more than 2.5 g albumin/mmolcreatinine for male patients or 3.5 g/mmol for female patients.Alternatively or in addition the albumin excretion rate can bedetermined in this case this rate should be below 20 or 30 mg/24 h formale or female patients respectively. As mentioned above, in thisembodiment, the method of the invention may be used with advantage toprovide a diagnostic and/or prognostic parameter for early kidney damageor the risk thereof, even before any such damage has progressed to sucha stage that microalbuminuria already occurs.

Furthermore, as already mentioned above, the invention may be applied todetect early changes in the peritoneal membrane as occurring ascomplication in CAPD. For this application of the invention, again aurine sample may be used, i.e. in the manner described above. However,as such patients may provide very little or no urine, this aspect of theinvention preferably involves the use of a dialysate fluid.

Accordingly, the invention also relates to a method for determiningwhether (a sample of) a dialysate fluid has been obtained from a subjectsuffering from (a disorder leading to) remodelling of the peritonealmembrane, said method comprising the steps of:

-   a) providing at least one sample of dialysate fluid from said    subject;-   b) determining in said at least one sample of dialysate fluid the    presence and/or the level of at least one proteolytic enzyme;    and optionally comprising the further step of:-   c) comparing one or more of the values determined in step b) with at    least one reference value or reference sample.

In particular, in this aspect of the invention, the (sample of)dialysate fluid will have been obtained from a patient that undergoes(or has undergone) dialysis, and in particular continuous ambulatoryperitoneal dialysis (CAPD).

The at least one proteolytic enzyme/protease determined in step b) ispreferably a protease involved in vascular remodelling. For instance, inthe invention, one or more proteases may be determined that are selectedfrom the group consisting of (matrix) metalloproteases and/or enzymesrelated to (matrix) metalloproteases. These include, but are not limitedto, the proteases schematically shown in FIG. 2, e.g.:

-   -   matrix metalloproteases (“MMPs”) such as MMP-1, MMP-2, MMP-3,        MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13; the        so-called “MT-MMPs” (e.g. MMP-14 to MMP-17); as well as MMP-18        and MMP-19-MMP-23; and/or    -   proteases related to MMPs such as ADAMs (e.g. ADAM-1 to ADAM-30)        or ADAM-TS (e.g. ADAM-TS 1 to ADAM-TS 7);        or any combination thereof.

Other suitable proteases will be clear to the skilled person based uponthe disclosure herein, also based upon the art relating to the above andsimilar proteases (vide for instance Massova et al., Primakoff et al.and Tang and Hong, supra). It is also envisaged that these may includeproteases which are only discovered after the priority date of thepresent application, e.g. based inter alia upon the teaching of thepresent application.

Preferably, in the method of the invention, the presence and/or thelevel(s) of at least one of the proteases MMP-1, MMP-2, MMP-8, MMP-9,MMP-13 and/or MMP-14 is determined, and in particular the presenceand/or the level(s) of MMP-2 and/or MMP-9; optionally in combinationwith the presence and/or the levels of one or more of furtherproteolytic enzymes, and in particular one or more of the furtherproteases mentioned above.

Even more preferably, the combination of proteases MMP-2 and MMP-9 isdetermined; again optionally in combination with the presence and/or thelevels of one or more of further proteolytic enzymes, and in particularone or more of the further proteases mentioned above.

Also, instead of the presence and/or the levels of one or more of theentire proteases mentioned above, the presence and/or the levels in aurine sample of one or more parts or fragments of such proteases may bedetermined. These may for instance be parts or fragments that may beconsidered representative for the presence of the corresponding proteaseand/or parts or fragments that are (also) indicative of kidney damage asdescribed herein. In particular, these may be (soluble) fragments ofnormally insoluble, membrane bound, enzymes which are still recognizableeither by remaining enzymatic activity or antigenic determinants. Suchfragments and the determination thereof should be consideredincorporated within the terms “proteolytic enzyme” or “protease” as usedherein.

Also, in step b), besides the presence and/or the levels of the one ormore of the proteases mentioned above, one or more other parameters ofthe urine sample may be determined, so as to provide additionalinformation to the clinician. These further parameters may for instanceinclude the presence and/or extent of microalbuminuria; the presenceand/or the level(s) of glucose/sugars, or the determination of thepresence of erythrocytes, leukocytes, bacteria, paraproteins, etc.

In the invention, when two or more proteases are to be determined, thesemay be determined in the same sample—which for this purpose may bedivided into several parts or fractions—or in different samples.Accordingly, the urine sample of step a) may be a single sample (or apart or fraction thereof) or may be a sample from a set of samplescollected from a patient, e.g. essentially simultaneously, for instanceon the same day, in the same week and/or at the same clinical stage. Itis also encompassed in the scope of the invention that initially onlyone or a limited number of the proteases mentioned above are determined,and that subsequently one or more further proteases are determined, forinstance to confirm the results of the initial determination and/or toprovide additional information.

The one or more value(s) obtained in step b) may also be compared to oneor more reference values. These may for instance be values obtained fromother samples of the same patient/individual (e.g. values obtained fromsamples collected earlier from the patient); values obtained using oneor more reference samples obtained from other patients or individuals(e.g. healthy individuals, individuals with kidney damage with orwithout albuminuria, and/or patients with diabetes); values obtainedthrough clinical practice or experience; and/or (average) valuesobtained from one or more groups of patients/individuals, for instanceusing statistical analysis. The latter may for instance be generated orcompiled over time using the method of the invention; it is however alsoenvisaged that after the priority date of the present application, theseor other reference values will become available as part of a manualand/or in the scientific literature.

Usually, any such reference value(s) will be—or will havebeen—determined by the same technique—e.g. using the same assay—as isused to determine the levels of the proteases in the sample of step a);although the invention in its broadest sense is not limited thereto.

The levels of the proteases in a given urine sample may be determined inany manner known per se, including but not limited to:

-   1) assay techniques involving conversion of natural or artificial    substrates followed by a physical separation method such as HPLC;-   2) assay techniques involving conversion of labelled natural or    artificial substrate followed by measurement of the released    (radioactive) label;-   3) assay techniques involving conversion of artificial fluorogenic    or chromogenic substrate.-   4) assay techniques involving zymographic detection in    substrate-containing gels;-   5) assay techniques involving conversion of modified pro-enzyme    substrate, in combination with immuno-capture;-   6) assay techniques involving detection of antigen by immunological    assay such as ELISA. or any combination thereof.

These methods may be carried out in a manner known per se, for instanceas described in the prior art mentioned above, in WO 97/41441, in EP 0691 409, as well as in D. E. Kleiner and W. G. Stetlerstevenson, Anal.Biochem. 218, 315-319 (1994). The method disclosed in EP 0 691 409 isparticularly preferred.

Kits for determining the proteases may also be commercially available.For instance, for methods 2) to 6) mentioned above, kits arecommercially available from Roche Diagnostics, Calbiochem, Novagen andAmersham Pharmacia Biotech (for both method 5 and method 6).

Thus, the assay(s) may also be carried out essentially according to theinstructions provided with these kits.

Preferably, an immunological method such as method 6 mentioned aboveand/or a functional method such as methods 1-5 mentioned above is used.

Most preferably, of the above methods 1) to 6), method 5) is used, asmethods 1) to 3) may be less or even non-specific; methods 1), 2) and 4)are generally laborious and not useable for large numbers of samples;and method 6) does not measure activity. Method 5) is also described inmore detail in EP 0 691 409.

Generally, each protease will be determined using a separate assayspecific for said protease (e.g. as described below). However, it isalso encompassed in the invention to use assays that can determine thepresence and/or the levels of two or more proteases, e.g. combinedand/or simultaneously. Also, each separate assay will be usually becarried out on a separate sample or a separate part or fraction of asample; although again the invention in its broadest sense is notlimited thereto.

In the invention, a sample obtained from a patient suffering from renaldamage or a renal disorder will show the presence of one or more of theproteases indicated above.

In particular, in the invention, a sample obtained from a patientsuffering from renal damage or a renal disorder will show, compared to asample obtained from a healthy individual, increased levels of one ormore of one or more of the proteases indicated above, e.g. increasedfrom a value of essentially zero to a measurable/detectable amount,and/or increased by at least a factor 2, in particular by a factor 5 orhigher.

More in particular, in the invention, a sample obtained from a patientsuffering from renal damage or a renal disorder will show one or more ofthe following:

-   -   the presence of MMP-2, and in particular the presence of        increased levels of MMP-2 compared to a sample obtained from a        healthy individual. For instance, the sample may show a urinary        MMP activity—e.g. as determined by method 5)—above of 200 U/mmol        creatinine;    -   the presence of MMP-9, and in particular the presence of        increased levels of MMP-9 compared to a sample obtained from a        healthy individual. For instance, the sample may show a urinary        MMP activity—e.g. as determined by method 5)—above of 200 U.mmol        creatinine;    -   the simultaneous presence of both MMP-2 and MMP-9 and in        particular the simultaneous presence of increased levels of        these enzymes as compared to a sample obtained from a healthy        individual.

Thus, the method of the invention may be used to determine whether agiven urine sample has been obtained from a patient suffering fromkidney damage or not; and thus may provide valuable information on theclinical status of said patient.

In addition, the method of the invention may for instance be used—afterit has been established (e.g. by the method of the invention or anyother method) that some (early) kidney damage has already occured—topredict and/or to follow the further course of such damage.

It is even envisaged that the method of the invention may even be usedto determine whether and/or when an individual—e.g. a healthy individualbut in particular a diabetes patient—is at (further increased) risk ofdeveloping renal damage. It may even be that the presence and/or thelevels of different proteases, and/or the ratio(s) of these levels, maybe indicative for different renal disorders and/or for different formsof renal damage; for different causes of renal damage; and/or fordifferent clinical stages of renal disorders or for a specific extent ofrenal damage.

Thus, the method of the invention provides the clinician with aconvenient yet very reliable and powerful tool for diagnosing,predicting and/or following renal disorders, renal damage and/or theclinical course thereof, in particular in patients that are at(increased) risk thereof. These may include but are not limited todiabetes patients; and more generally comprise patients of any diseaseand/or disorder that may cause and/or lead to kidney damage (also as acomplication of said disease and/or disorder). For instance, the resultsobtained using the method of the invention may lead the clinical toprescribe treatment or to change or modify a treatment alreadyprescribed.

Also, method of the invention may be used in diagnosing, predictingand/or following (early) kidney renal in subjects that are undergoingtreatment with any medication that may cause kidney damage (e.g. as aside-effect of said medication). Again, the results obtained using themethod of the invention may lead the clinical to change or modify saidtreatment.

It is also envisaged that, besides the above clincal application(s), themethod of the invention may also find applications in other areas, forinstance to determine whether a new drug may cause kidney damage (e.g.as a side-effect) and/or in studying the effects of drugs that areintended to prevent and/or counteract kidney disorders and/or kidneydamage, e.g. damage caused by dysregulation of remodelling processes.Thus, it is expected that the method of the invention may also be usefulfor research purposes, for instance in drug discovery, drug testingand/or drug development.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to determine the level of a protease or a numberof proteases in urine as a diagnostic, prognostic or otherwise medicallyinteresting marker for the occurrence, progression, therapeutic effector otherwise medically interesting fact of diseases possibly leading tovascular complications in general and more specifically to damage to the(micro) vasculature of the kidney resulting in partial, gradual, acuteor total loss of function of this organ. More particular the presentinvention relates to (very) early detection of processes eventuallyleading to such a damage. Ideally this detection is early enough to beable to prevent serious damage by timely starting a suitable therapy orpreventive strategy.

The phrase “determining the level of a protease or a number ofproteases” means both qualitative analysis i.e. determining the presenceof the protease or proteases above a certain well chosen threshold leveland quantitative analysis i.e. determining the level of proteasesbelonging to the family of matrix-metalloproteases (MMPs), ADAMs orADAMTSs (table I). This is not limited to only those proteases sincevirtually any protease involved in vascular remodelling might serve thepurpose in certain cases, conceivably dependent on the specificpathology leading to the kidney damage. Particularly interesting are thevarious MMPs (table 2), enzymes known to be involved in local tissueremodelling and degradation.

Detection or quantification of the particular protease or proteases canbe based on its enzymatic activity, or alternatively based on detectionof the antigen. For both ways of detection general or specificmethodology is available or can be devised based on currently knowntechnology (table 3). It is conceivable that detection or measurement ofa combination of two or more proteolytic enzymes is a way to obtainsufficient reliability. The method focuses on urine as a biologicalfluid since in urine of healthy control subjects hardly any proteolyticenzyme can be detected and urine is currently used as a biological fluidfor diagnostic and other medical purposes in e.g. the field of diabetesand consequently fits well with current medical and clinical chemistrypractice.

Possible applications of the present invention include, but are notlimited to:

-   -   (early) diagnosis of renal damage resulting from diabetes or        other diseases, enabling the start of therapeutic or preventive        actions before renal failure occurs;    -   monitoring the effect of therapeutic or preventive intervention;    -   prediction of the most likely progression of the disease related        renal damage; more specifically damage to the microvasculature        of the kidney frequently occurs as a complication of diabetes        mellitus but can also have other causes.

Early detection is important to prevent serious damage leading to kidneyfailure. At present the determination of trace quantities of albumin inthe urine (microalbuminuria) is the established method to detect renaldamage. A method that would give an earlier warning for still initialand possibly reversible renal damage would be desirable.

Thus, the invention provides a non-invasive method for facilitatingdiagnosis or prognosis of kidney damage in a subject comprisingobtaining a sample of urine from said subject and detecting aproteolytic enzyme in said sample thereby facilitating the diagnosis ofthe subject for conditions leading to kidney damage, in which theproteases and the manner in which these proteases are determined are asmentioned above. The condition leading to kidney damage may inparticular be diabetes, more in particular be diabetes type 1 and/ordiabetes type 2.

In another aspect, the invention also relates to the use of an assay fordetermining the presence and/or the level of at least one protease asmentioned above, in determining whether a urine sample has been derivedfrom a patient suffering from a kidney disorder and/or kidney damage,and in particular from kidney damage caused by diabetes, in which saidassay is used to determine the presence and/or the levels of said atleast one protease in said urine sample. In particular, as mentionedabove, the urine sample is a urine sample obtained from a patient thatis (judged to be) at risk of—and/or that suffers from and/or issuspected to suffer from—such kidney damage, and/or may be a urinesample obtained from a patient suffering from diabetes.

In another aspect, the invention relates to the use of means fordetermining the presence and/or levels of at least one proteolyticenzyme, in a method as described above. Such means may for instance bemeans for carrying out one of the assay techniques referred to above,such as the methods 1) to 6). Usually said means will be (provided tothe end-user) in the form of a kit, and such kits are known in the artand may be commercially available.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph/diagram showing total (active and activatable) MMP-9in 24-h urine collections of type-I diabetic patients with albumin inurine (numbers 1-16) and without albumin in urine (numbers 17-30), asdetermined with a commercially available immunocapture assay kit forMMP-9 activity (Amersham Pharma Biotech RPN 2630) and as expressed asunits per mmol creatinine;

FIG. 2 is a graph/diagram showing total (active and activatable) MMP-2in 24-h urine collections of type I diabetic patients with albumin inurine (numbers 1-16) and without albumin in urine (numbers 17-30), asdetermined with a commercially available immunocapture assay kit for MMP-2 activity (Amersham Pharma Biotech RPN 2631) and as expressed as unitsper mmol creatinine;

FIG. 3 is a graph/diagram showing total MMP-9 activity in urine ofpatients with rheumatoid arthritis before and after treatment with adrug supposed to inhibit cartilage degradation, as determined with acommercially available immunocapture assay kit for MMP-9 activity(Amersham Pharma Biotech RPN 2630) and as expressed as units per mmolcreatinine.

FIG. 4 is a graph/diagram showing total MMP-2 and MMP-9 activity inurine of type I diabetes patients with microalbuminuria. Activities weremeasured with commercially available immuno capture kits (AmershamPharmacia Biotech RPN 2631 and RPN 2630).

FIG. 5 is a graph/diagram showing total MMP-2 and MMP-9 activity inurine of type I diabetes patients without microalbuminuria. Activitieswere measured with commercially available immuno capture kits (AmershamPharmacia Biotech RPN 2631 and RPN 2630).

EXAMPLES Example I Measurement of MMP-2 Antigen in Serum of HealthyControls and Diabetic Patients with and without Albuminuria.

The level of matrix-metalloprotease-2 (MMP-2) antigen in serum ofhealthy control (n=9) and patients with type I diabetes with (n=16) andwithout (n=140) albuminuria was determined using a commerciallyavailable ELISA (Amersham Pharmacia Biotech RPN 2617 kit).

No significant differences in levels between the various groups werefound: control 547±38 ng/ml, diabetes patients with albuminuria 608±42ng/ml and diabetes patients without albuminuria 612±44 ng/ml (mean±SEM).

Example II Measurement of MMP-9 Antigen in Serum of Healthy Controls andDiabetic Patients with and without Albuminuria.

The level of matrix-metalloproteases-9 (MMP-9) in serum of healthycontrols (n=9) and patients with type I diabetes with (n=16) and without(n=14) albuminuria was determined using a commercially available ELISA(Amersham Pharmacia Biotech RPN 2614 kit). Patient serum MMP-9 levelswere significantly increased versus controls (508±38 ng/ml vs 159±17ng/ml; p<0.001). No difference was observed between patients with andwithout albuminuria.

Example III Inactive Pro-Forms of MMPs are Converted to Active Forms byTreatment with Amino-phenyl-mercuric-acetate (APMA).

A MMP containing sample after immunocapture is incubated with 0.5 mMAPMA at 37 C for 2 h (MMP-9) or 0.5 h (MMP-2) as described in theinstructions of the manufacturer of the kits. Measurement without APMAtreatment gives active MMP present in the sample, whereas measurementafter APMA treatment gives active and activatable pro-enzyme in thesample.

Example IV Measurement of MMP-2 Activity in Urine of Healthy Controlsand Diabetic Patients with and without Albuminuria.

Total Active and activatable MMP-2 was determined in 24-h urinecollections of controls and patients with a commercially availableimmunocapture assay kit for MMP-2 activity (Amersham Pharma Biotech RPN2631) and expressed as units per mmol creatinine. MMP-2 level incontrols was non detectable (<0.1 units/mmol creatinine), whereas urineof diabetic patients with albuminuria had significantly increased levelsas compared with patients without albuminuria (median 458) (range0-10959) versus 0 (range 0-3026) units/mmol creatinine (p <0.003 MannWhitney U test). (see FIG. 2).

Example V Measurement of MMP-9 Activity in Urine of Healthy Controls andDiabetic Patients with and without Albuminuria.

Total and activatable MMP-9 levels were determined in 24h urinecollections of healthy controls and diabetic patients using acommercially available immunocapture assay kit for MMP-9 activity(Amersham Pharmacia Biotech RPN 2630). Total (active +activatable) MMP-9was not detectable in urine of normals, whereas it was clearly increasedin urine of diabetic patients with albuminuria versus patients withoutalbuminuria (median 964 (range 0-26129) versus 0 (range 0-3519)units/mmol creatinine). (See FIG. 1).

Example VI Measurement of total MMP-9 Activity in Urine of Patients withRheumatoid Arthritis before and after Treatment with a Drug supposed toinhibit Cartilage Degradation.

MMP-9 activity was measured with the method of example V. (See FIG. 3).

Example VII Measurement of total MMP-2 and MMP-9 Activity in Urine ofPatients with Microalbuminuria or Normoalbuminuric Patients.

In patients with microalbuminuria at least one of the two measured MMPsis detectable (see FIG. 4), whereas in normoalbuminuric patients neitherof the two, or sporadically one of the two MMPs measured is detectable(see FIG. 5).

1. A method for determining whether a urine sample has been obtainedfrom a subject having a kidney disorder, wherein a test of said urinesample indicated that the subject does not exhibit microalbuminuria, themethod comprising the steps of: a) obtaining a urine sample from thesubject; b) determining in the urine sample the levels ofmetalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2); c)comparing said levels of MMP-9 and MMP-2 determined in step b) withreference values, and d) determining whether the subject has a kidneydisorder based on the presence of increased levels of MMP-9 and/or MMP-2compared to the reference values.
 2. A method for determining whether aurine sample has been obtained from a subject having a kidney disorderwherein the subject has diabetes type I or diabetes II, wherein a testof said urine sample indicated that the subject does not exhibitmicroalbuminuria, the method comprising the steps of: a) obtaining aurine sample from the subject; b) determining in the urine sample thelevels of MMP-9 and MMP-2; c) comparing one or more of the valuesdetermined in step b) with reference values or reference samples, and d)determining whether the subject has a kidney disorder based on thepresence of increased levels of MMP-9 and/or MMP-2 compared to thereference values or reference samples.
 3. The method according to claim2, wherein said subject has type I diabetes.
 4. The method according toclaim 2, wherein said subject has type II diabetes.
 5. A method fordetermining whether a urine sample has been obtained from a subjecthaving a kidney disorder, the method comprising the steps of: a)obtaining a urine sample from a subject, b) determining in the urinesample the levels of MMP-9 and MMP-2; c) comparing said levels of MMP-9and MMP-2 determined in step b) with reference values, and d)determining whether the subject has a kidney disorder based on thepresence of increased levels of MMP-9 and/or MMP-2 compared to thereference values.
 6. The method according to claim 5, wherein said urinesample indicated that the subject does not exhibit microalbuminuria.